This invention relates generally to the in vitro analysis, using a dry test strip, of plasma, serum or whole blood samples, and more specifically, to assay for cholesterol from Low Density Lipoproteins (LDL-C) contained in samples.
The level of cholesterol in blood has become accepted as a significant indicator of risk of coronary heart disease. Cholesterol is contained and is transported in lipoproteins in blood. “Total Cholesterol” includes cholesterol from Low Density Lipoproteins (LDL-C), from Intermediate Density Lipoproteins (IDL-C), from Chylomicrons, from Very Low Density Lipoproteins (VLDL-C) and from high density lipoproteins (HDL-C). It is well established from epidemiological and clinical studies that there is a positive correlation between levels of LDL-C and to a lesser extent of Lp(a)-C to coronary heart disease. Traditionally, LDL-C has been identified as “bad” cholesterol. On the other hand, clinical studies have established a negative correlation between levels of HLD-C (“good” cholesterol) and coronary heart disease. Standing alone, the level of total cholesterol in blood, which is a measure of the sum total of HDL-C, LDL-C, IDL-C, VLDL-C and Chylomicrons-C, is not generally regarded as an adequate indicator of the risk of coronary heart disease because the overall level of total cholesterol does not reveal the relative proportions of cholesterol from these sources. To better assess the risk of heart disease, it is desirable to determine the amount of LDL-C in a sample in addition to the total cholesterol in the sample.
The most common approach to determining LDL-C in the clinical laboratory is the Friedewald calculation, which estimates LDL-C from measurements of total cholesterol, HDL-C and triglycerides. Although convenient, the Friedewald calculation suffers from several well-established drawbacks. Nauck et al. “Methods of Measurement of LDL-Cholesterol: A critical Assessment of Direct Measurement by Homogeneous Assays versus Calculation” Clin. Chem. 48.2 (2002). For example, because the Friedewald calculation involves measurother than LDL-C, it is subject to potential compounded inaccuracies from the determinations of the other lipids in the equation. Further, its usefulness is known to be limited to biological fluids with trigylceride levels below 400 mg/d L, and its accuracy reportedly declines with triglyceride levels greater than 200 mg/dL.
Ultra-centrifugation is a known technique to separate and to quantify the various lipoprotein components from serum or plasma samples. However, ultra-centrifugation is tedious, time consuming, and the highly labile lipoproteins can be substantially altered by the high salt concentrations that are a part of the ultra-centrifugation process as well as by centrifugal forces. “Furthermore, a plethora of different types of equipment and tubes are used, making conditions difficult to reproduce from one laboratory to another and consistent separations highly dependent on the skills and care of the technician.” Id. At 238.
Another technique for measuring LDL-C is electrophoresis. This technique also has certain drawbacks. Electrophoresis gel assays do not lend themselves readily to automation and their accuracy and repeatability depends at least in part on the technique of the technician performing the test.
Other so-called homogeneous methods that involve precipitation of non-LDL lipoproteins, heating and additional steps, have recently become available. One homogeneous method for determining LDL-C is disclosed in U.S. Pat. No. 5,888,827 (Kayahara, Sugiuchi, et al.; assigned to Kyowa Medex Co., Japan). The '827 patent describes a two-stage liquid phase reaction to quantify LDL-C concentration in a fluid sample. In the first step, the sample containing LDL-C is placed in a first reagent that includes trimethyl beta-cyclodextrin as a sugar compound, polyoxyethylene monolaurate as a protein solubilizing agent, EMSE (N-ethyl-N-(3-methylphenyl)-N′,succinylethylenediamene) and Tris buffer. The reaction mixture is then heated to 37° C., and after 5 minutes the absorbance is read. A second reagent including cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine and Tris buffer is then added and after another 5 minutes the absorbance is again measured at the same wavelength. LDL-C is then calculated by separately subjecting a standard solution of cholesterol to the same procedure and comparing the respective absorbance values. For many applications the manipulations required in the practice of this method such as heating, multiple reagents and multiple readings is considered a drawback. Because this method is complex and tedious to perform even in a laboratory, it would not be suitable for a point-of-care (POC) environment.
Another two-stage homoegneous assay is disclosed in U.S. Pat. No. 6,194,164 (Matsui et al.; assigned to Denke Seiken, Ltd. Japan). In the first stage, HDL-C, VLDL-C and Chylomicron-C in the test sample are eliminated and, in the second step, the cholesterol remaining in the test sample (viz., LDL) is quantified. In the first step, cholesterol esterase and cholesterol oxidase act on the test sample in the presence of a surfactant that acts on lipoproteins other than LDL-C (“non-LDLs”). The hydrogen peroxide thereby generated is decomposed to water and oxygen by catalase. Alternatively, a phenol-based or aniline-based hydrogen donor is reacted with the hydrogen peroxide to convert it to a colorless compound. Preferred surfactants that act on the non-LDLs include polyoxyethylene laurl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, and the like. In the second reaction disclosed in the '164 patent, cholesterol remaining in the test sample, which should theoretically contain only LDL-C, is quantified. The second step may be carried out by adding a surfactant that acts on at least LDL and quantifying the resulting hydrogen peroxide by the action of the cholesterol esterase and the cholesterol oxidase added in the first step.
As with the method disclosed in the '827 patent, one disadvantage of the method taught by the '164 patent is that it requires heating the reaction mixture to a temperature of 37° C., and experimental data indicates that the test accuracy suffers at lower temperatures. Also as taught in the '827 patent, the method of the '164 patent requires multiple reagents to be added at different times, making it equally incompatible with POC testing or use in over-the-counter (“OTC”) applications.
A homogeneous assay for measuring LDL-C in serum was disclosed by H. Sugiuchi et al., Clinical Chemistry 44:3 522-531 (1998. This disclosure shows a correlation between the use of a combination of triblock copolymer and alpha-cyclodextrin sulfate and the selective enzymatic reaction of LDL-C when both LDLs and non-LDLs are contacted with the combination in a liquid assay system. The preferred polyoxyethylene-polyoxypropylene block copolymer of the Sugiuchi et al. disclosure exhibited limited solubility under liquid assay reaction conditions, rendering the adaptation to a dry strip unworkable.
Co-pending and commonly assigned U.S. patent application Ser. No. 10/663,555, filed Sep. 16, 2003, discloses a one-step, room-temperature whole blood, dry chemistry assay for LDL-C in which the amount of LDL-C present in whole blood is calculated from the results of direct measurements of total cholesterol and non-LDL-C. Although the disclosed assay overcomes most of the problems of the multi-step, wet chemistry LDL cholesterol assays of the prior art, there remains a preference for direct assays. Thus, there remains a need for a convenient, easy to use, dry, one-step, room-temperature diagnostic test for directly measuring LDL-C.